Experimental Study Of SHH Gene-modified Nasal Mucosal Mesenchymal Stem Cells In The Treatment Of Spinal Cord Injury In Rats

Spinal cord injury(SCI)is the most serious complication of spinal fracture,which not only has a serious impact on the patient and the family,but also imposes a heavy burden on society.In recent years,the incidence of SCI has been increasing year by year.After spinal cord injury,the fracture and collapse of the upper and lower nerve conduction bundles are the direct cause of limb sensation and motor dysfunction below the injury plane.After the spinal cord injury,secondary inflammatory reaction,then tissue necrosis,and finally form a cavity,The scar tissue formed by the proliferation of glial cells will hinder the regeneration of nerve fibers,eventually leading to necrosis and degeneration of neurons and nerve fibers.Ectomesenchymal stem cells(EMSCs)are characterized by simple changes,easy isolation and culture in vitro,and can be provided by donors without involving ethical issues.They are ideal seed cells for cell transplantation.In the treatment of spinal cord injury,EMSCs of the nasal mucosa can secrete cell active factors and extracellular matrix,which can improve the microenvironment of spinal cord injury,fill the necrotic cavity,bridge the spinal cord end,restore the anatomical structure,and guide the regeneration of endogenous nerve fiber network.However,due to changes in the local microenvironment after spinal cord injury,a large number of cells did not survive well after transplantation of nasal mucosal EMSCs.Sonic hedgehog(SHH)is a factor produced by the notochord during embryonicdevelopment.SHH determines the development and differentiation of the nervous system.In vitro studies have shown that SHH can induce adult stem cells to differentiate into motor neurons and oligodendrocytes,and SHH can promote neuronal survival and axonal regeneration.However,the SHH state in the body is unstable and will be quickly cleared by the body.In this experiment,we constructed SHH adenovirus transfected into nasal mucosa EMSCs.In vitro data showed that SHH adenovirus transfected EMSCs in nasal mucosa still maintained good biological characteristics and stably secreted SHH.At the same time,we found that SHH-modified nasal mucosa EMSCs were more likely to differentiate into neuron-like cells;then we transplanted SHH gene.Modified nasal mucosa EMSCs cells are used to treat spinal cord injury in rats,and try to solve the problem of rapid clearance of SHH and survival of nasal mucosa EMSCs.This method can effectively promote the recovery of function after spinal cord injury.This experiment has carried out related work from the cellular level and the animal level.The research content mainly includes the following four parts:1 In vitro isolation and culture of ectomesenchymal stem cells and culture and identification.The healthy Sprague Dawley(SD)rats were taken out,the nasal septum was removed under sterile conditions,the nasal mucosa was removed,and the cells were fully disrupted and placed in a DMEM/F12 containing 10% fetal bovine serum,a cell culture incubator at 37 ° C,5% CO2,the morphology and growth of the adherent cells were observed under an inverted microscope,then take pictures.The third generation was inoculated into a 96-well culture plate at 1,3,5,and 7 days,and the MTT method was used to detect the light absorption value of each well at a wavelength of 490 nm,and the results were recorded.The third generation of well-grown nasal mucosal mesenchymal stem cells were inoculated into 48-well plates,and the surface markers of nasal mucosal mesenchymal stem cells were detected by immunofluorescence,including: Nestin,Vimentin,Connexin-43 and CD133;Nasal murine mesenchymal stem cells were seeded in a 6-well cell culture plate,and an osteoblast inducer and an adipocyte inducer were added,and a blankV control was set.The results showed that the morphology of rat nasal mucosal mesenchymal stem cells was mainly fish-like,and the cells cultured in vitro could be stably transferred to the 10 th generation;the growth curve showed that the cells isolated and cultured were active;the cells obtained by immunofluorescence showed high expression of Nestin.Vimentin,Connexin-43 and CD133;after osteogenic and adipogenic differentiation,both alizarin red staining and oil red O staining were positive.2 In vitro construction of SHH adenovirus transfected nasal mucosal mesenchymal stem cells and identificationThe SHH adenovirus construction technology was supported by Changsha Yingrun Technology Co.,Ltd.;SHH adenovirus was transfected into nasal mucosa EMSCs,and the transfection efficiency was determined by fluorescence expression;the intracellular and culture of SHH-EMSCs group and EMSCs group were determined by western-blot method.The expression of SHH protein in liquid supernatant;the differentiation of nasal mucosa EMSCs induced by SHH gene into osteoblasts and adipocytes.The results showed that the expression of green fluorescent protein was significantly enhanced in the transfected EMSCs transfected with SHH recombinant adenovirus vector for 48 h after culture.The SHH gene-modified nasal mucosal mesenchymal stem cells could still differentiate into osteoblasts and adipocytes.3 In vitro induction of SHH gene-modified nasal mucosal mesenchymal stem cells differentiate into neuron-like cellsThe third-generation nasal mucosa EMSCs with good growth were used for the experiment.The experiment was divided into four groups: SHH adenovirus transfection induction group,SHH adenovirus transfection group,and blank control group(normal nasal mucosa EMSCs induced and uninduced group).Different groups of cells were induced to differentiate into neuron-like cells,and the morphology of each group was observed by an inverted microscope.Immunofluorescence and immunoblotting were used to detect the expression of neuro-related proteins and expression of proteins related to the downstream of SHH signaling pathway in each group.The results showed that after induction,the cells in the SHH transfection grouphad morphological changes of the fish-like cells to the typical neuron-like cells,and the expression levels of nerve-related proteins were higher than those of the other three groups.4 Observation of SHH gene-modified nasal mucosal mesenchymal stem cells for repairing spinal cord injury in ratsSPF adult SD rats were taken and a spinal cord injury model was established.Divided into two parts,the first part transplanted SHH-EMSCs cells into the spinal cord injury.After 10 days of feeding,the spinal cord specimens were taken for immunofluorescence staining to observe the survival and migration.In the second part,SD rats were randomly divided into the following groups:(A)SHH-EMSCs transplantation group;(B)EMSCs transplantation group;(C)simple transection group.Eight animals were used in each group and kept for 10 weeks.Rats were anesthetized with 1% sodium pentobarbital solution,T10 was used as a marker,laminectomy was performed,spinal dural sac was exposed,and the spinal cord was transected;nasal mucosal mesenchymal stem cells were transplanted after adequate hemostasis.Ten weeks after transplantation,removed the spinal cord tissue and detected the levels of NF200 and TUBB3 protein in spinal cord injury by Western blot.HE and immunohistochemistry were performed by frozen section of injury center.The results showed that SHH-EMSCs survived and differentiated into TUBB3 positive cells in rat spinal cord injury.After ten weeks,rats in the SHH-EMSCs group were significantly better than the other two groups in recovery of motor function.